Regulation of nitric oxide synthase activity in cultured human endothelial cells: effect of antioxidants

Nitric oxide release is induced in many cells, including vascular endothelium, as part of the host response to inflammation. Nitric oxide synthase activity is increased in patients with sepsis, associated with increased oxidant demands and decreased antioxidant protection. We used a human vascular endothelial cell line to investigate the influence of antioxidants on nitric oxide synthase activity. Cells were cultured to confluence and incubated with interferon gamma, tumor necrosis factor, and lipopolysaccharide in the combined presence of the antioxidants ascorbic acid, Trolox, catalase, or superoxide dismutase, singly and in combination, for 48 h. Additionally, some cells were incubated with hypoxanthine-xanthine oxidase or a nitric oxide donor. Nitric oxide synthase activity was upregulated by cytokine exposure (p < .0005). Ascorbic acid and superoxide dismutase/ catalase resulted in decreased enzyme activity (p < .05). Superoxide anion release from xanthine oxidase caused increased activity (p < .05) and exogenous nitric oxide tended to suppress synthase activity. We suggest that antioxidants scavenge superoxide anion, enabling feedback inhibition of nitric oxide synthase activity by nitric oxide, and thus reducing enzyme activity. Exogenous nitric oxide also has a similar effect. Superoxide generation suppresses this feedback inhibition. This study has important implications in patients with sepsis in whom nitric oxide synthase inhibitor therapy is currently under investigation.